The goal of this project is to provide information about the nature of pre-mRNA splicing in Schizosaccharomyces pombe (fission yeast). This goal will be accomplished by developing and characterizing an in vitro system in which exogenously added mRNA precursors are spliced in the presence of S. pombe extracts prepared from spheroplasts. The mRNA precursors will be generated by in vitro transcription of linearized plasmids which contain gene segments with introns (SV40 early gene and others) fused to the bacteriophage T7 RNA polymerase promoter. Analysis for spliced RNAs will be accomplished by S1 protection assays and RNA sequencing. The in vitro system will be utilized to assess the kinetics of the reaction and the role of monovalent and divalent cations, nucleotides, temperature, RNA substrate concentration, extract concentration, pH, and snRNPs. Mechanisms of splicing in the system will be investigated by characterizing potential linear and nonlinear RNA splicing kinetic intermediates. If present, the latter species will be identified by two-dimensional gel electrophoresis. Analysis for the possible presence of lariat structures will be accomplished by S1 nuclease- mapping, primer extension, susceptibility to lariat debranching enzyme, and RNase P1 digestion analysis. Utilizing the in vitro system, formation of spliceosomes will be investigated by glycerol gradient centrifugation and native gel electrophoresis. Complexes will be analyzed for the presence of pre-mRNA, intermediates, and/or spliced product. The presence of snRNAs in spliceosomes will be assessed by the analysis of RNAs from gel- resolved complexes which are immunoprecipited by anti-2,2,7- trimethylguanosine cap antibodies.